This project seeks support for exploring novel physical principles from which new methodologies appropriate to mapping and sequencing megabase sized DNA molecules can be developed. Recently, my group has shown that combining direct observation of individual DNA molecules under fluorescence microscopy with physical principles of coil relaxation makes it possible to produce restriction maps of large molecules rapidly and with high resolution using single molecules. Since individual molecules are resolved in the microscope, the problem of polydispersity which besets standard hydrodynamic measurements is bypassed. Extension of this approach to optical sequencing of molecules, based on measurement of rotational diffusion constants under the microscope, is possible theoretically, and I proposed to work out the experimental details. Together these methods offer a powerful and versatile approach to genomic analysis that is easily and directly automatable. We propose to investigate the experimental variables and practical details of the image analysis required to develop single molecule optical methodology into a practical analytical system. The detailed mechanisms underlying the response of very large DNA's to orienting electrical pulses will be investigated systematically, in an effort to extend the resolution of electrophoresis so that separation of mammalian chromosome sized molecules can be achieved. Success of this program has the potential to accelerate mapping and sequencing of megabase sized DNA's by orders of magnitude over current procedures.